Project Summary/Abstract Stem cells, including pluripotent stem cells (PSCs) and tissue-specific stem cells, can differentiate into functional tissues and therefore serve as promising donor sources for cell-based therapies. Mouse models have played indispensable roles in understanding the basic biological features and potential applications of stem cells. However, because human and rodent have diverged from a common ancestral species over 85 million years ago, it is not surprising that human stem cells also possess features that are not conserved in mouse cells. Therefore, an improved understanding to the similarities and differences between human and mouse stem cells is critical for using mice to model human diseases and test stem cell therapies. MicroRNAs (miRNAs) play a central role in cell proliferation, differentiation, and survival. Mouse PSCs and neural stem cells (NSCs) deficient for Dicer or Dgcr8, which encode factors essential for miRNA biogenesis, can self-renew but cannot differentiate, demonstrating that miRNAs are not essential for self-renewal of these mouse cells. Because how miRNAs regulate human stem cells have not been systematically investigated, whether similar miRNA regulation is conserved across species remains unclear. In the preliminary studies, we generated DGCR8flox/flox human PSCs (hPSCs), which enable DGCR8 inactivation in PSCs and the differentiated progeny. Our preliminary data showed that, different from mouse cells, miRNAs are required for self-renewal of human PSCs and NSCs. Furthermore, self-renewal is impaired in hPSCs with only one functional DGCR8 allele. Importantly, we identified that miR-302 or miR-92, when expressed alone, is sufficient to rescue self-renewal of DGCR8-/- human PSCs or NSCs, respectively. Based on these data, we hypothesize that self-renewal of human PSCs and NSCs require miRNAs such as miR-302 or miR-92, which differ fundamentally from the mouse stem cells. The rationale is that knowledge on how miRNAs regulate human stem cells will improve our understanding to the differences between human and mouse stem cells, which will allow recapitulation of human diseases in mouse models with greater precision and make the mouse models more predictable, usable, and applicable for biomedical research. Aim 1 will compare how miR-302 regualtes human and mouse PSCs, Aim 2 will compare how miR-92 regulates human and mouse NSCs, and Aim 3 will compare how heterozygous loss of DGCR8 affects human and mouse PSCs and NSCs. The expected outcomes are to 1) discover the fundamental differences regarding to miRNA regulation between human and mouse stem cells, which will be critical for the use of mouse models for stem cell research and regenerative medicine applications; and 2) gain knowledge on how miRNAs regulate human stem cells, which will serve as the basis for future comparison with stem cells from other animal models.